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Oral Presentation at NCI
- 1. Locating regions in Dnak important for chaperone activity Roxana Hernandez 7/27/12 DNA Molecular Laboratory, NCI,NIH
- 2. DnaK • Source from Escherichia coli • DnaK/Hsp70 (70 kilodalton heat shock proteins) with similar structure exist in almost all living organisms • Important for protein folding and to help protect cells from stress • Functions with co- chaperones
- 3. Structure of DnaK ATP ATPase domain peptide Substrate- binding domain Peptide bound model - Stevens, S.Y. et al., (2003) Protein Science Nucleotide bound model - Sousa, M.C. and McKay, D.B (1998) Biochemistry N- and C-terminal combined model based on Hsc70 structure - Jiang, J. et al., (2005) Molecular Cell
- 5. Goals • Inhibiting molecular chaperones involved in activating oncogenic proteins could help cure cancer
- 6. Methods Mutated plasmids transformed into cells for growth using ‘QuickChange’ for Site- directed mutagenesis Collected DnaK 351 mutated plasmid for sequencing If mutation is correct then the plasmid is used for protein overproduction
- 7. Methods • Protein prep: ① Grow culture (overproduction of proteins) ② Lyse cells using a French Press ③ Column Chromatography kDa 181.8 82.2 64.2 48.8 37.1 25.9 19.4 M P S P S U I 14.8
- 8. Column Chromatography Q-Sepharose Anion Exchange S100 Size Exclusion kDa 181. 8115.5 82.2 37.1 48.8 25.9 19.4 64.2 70 D L W M D M
- 9. Future Plans Monitor disaggregation by measuring the increase in fluorescence Native Green Fluorescent Protein (GFP) Heat denature Incubate with chaperones & ATP Refolded GFP Heat-aggregated non-fluorescent GFP
- 10. Significance • Investigating the role of chaperones in diseases of protein misfolding • Inhibiting the chaperone involved could help cure disease – Ex: Cancer, Cystic Fibrosis, Sickle Cell, Type II Diabetes..
- 11. Acknowledgements • Sue Wickner • Shannon Doyle • Joel Hoskins • Danielle Johnston • Olivier Genest • Colleen Berringer DNA Molecular Laboratory, NCI,NIH
Editor's Notes
- Dnak 351 mutant on an overproduction plasmid Site-directed mutagensis Move mutated plasmids into cells and grow Plasmid prep—collect mutated plasmid Sequence mutated plasmid If sequence has correct mutation move into cells for protein production
- Grow large culture (overproduce protein) Lyse cells using french press Separate proteins from cell debris (pellet form) Anonic exchange column (Q-sepharose column) Gel filtration (S100 Column) size exclusion M-Marker P-Pellet S-Supernatant U-Uninduced I-induced
- You should do an arrow that goes from the Q18 in Q-seph and Brackets to the Sfractions **Gels from Columns S100 & Q-sepharose Anion Exchange= Q-Sepharose Columns Size Exclusion= S100 Columns D=dnaK Protein Supernatant M=Marker kDa Supernatant is tested for the presence of the target protein and checked for purity **S14 should have been a little more than that it fell out